Methods and systems for moving fluid in a microfluidic device

ABSTRACT

The present invention relates to a system and method for moving samples, such as fluid, within a microfluidic system using a plurality of gas actuators for applying pressure at different locations within the microfluidic. The system includes a substrate which forms a fluid network through which fluid flows, and a plurality of gas actuators integral with the substrate. One such gas actuator is coupled to the network at a first location for providing gas pressure to move a microfluidic sample within the network. Another gas actuator is coupled to the network at a second location for providing gas pressure to further move at least a portion of the microfluidic sample within the network. A valve is coupled to the microfluidic network so that, when the valve is closed, it substantially isolates the second gas actuator from the first gas actuator.

RELATED APPLICATIONS

[0001] This application is a continuation-in-part of application Ser.No. 10/014,519, filed Dec. 14, 2001. This application is also acontinuation-in-part of application Ser. No. 09/953,921, filed Sep. 18,2001, and claims priority of provisional application No. 60/307,638filed Jul. 26, 2001. This application is also a continuation-in-part ofapplication Ser. No. 09/819,105, filed Mar. 28, 2001. Each of theabove-mentioned applications are incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The present invention relates to methods and systems forprocessing samples using microfluidic systems. More particularly, theinvention relates to moving fluid samples within a microfluidic system.

BACKGROUND

[0003] Microfluidic devices are typically formed of substrates (made ofsilicon, glass, ceramic, plastic and/or quartz) which include a networkof micro-channels through which fluid flows under the control of apropulsion mechanism. The micro channels typically have at least onedimension which is on the order of nanometers to hundreds of microns.

[0004] Microfluidic devices process minute amounts of fluid sample todetermine the physical and chemical properties of the sample.Microfluidic devices offer several advantages over a traditionalmacro-scale instrumentation. For example, in general, they requiresubstantially smaller fluid samples, use far less reagent, and processthese fluids at substantially greater speeds than macro-scale equipment.

[0005] Electric fields are used as a propulsion mechanism for somemicrofluidic devices. In such devices, a high voltage, on the order ofkilovolts, is applied across electrodes within the device to therebygenerate an electric field in the micro channels. The field imposes aforce on ions within the fluid, thereby propelling the ions through themicro channel. The fluid itself may also be propelled by the motion ofions moving within the fluid.

[0006] Gas pressure is also used to propel fluid through micro channels.In some devices, a source of pressurized gas, external to themicrofluidic device, is connected to the microfluidic device to supply agas pressure, which propels the fluid. Gas pressure may also begenerated by a heated chamber within the microfluidic device itself topropagate fluid within a micro channel.

SUMMARY OF THE INVENTION

[0007] In general, the invention relates to a system and method formoving samples, such as fluids, within a microfluidic system. In oneaspect, the invention relates to the use of a plurality of gas actuatorsfor applying pressure at different locations within the microfluidicsystem to thereby supply force for moving samples. For example, in oneembodiment, a first gas actuator provides a gas pressure sufficient tomove a first sample from a first location to a second location of themicrofluidic device. A second gas actuator provides a gas pressure tomove another sample from a third location to a fourth location of themicrofluidic device.

[0008] In another example, a plurality of gas actuators cooperate tomove the same fluid sample. A first gas actuator provides a gas pressuresufficient to move the microdroplet between first and second processingzones of the microfluidic device, and a second gas actuator provides agas pressure to move the microdroplet to a third processing zone.

[0009] In preferred embodiments, the plurality of actuators are integralwith a microfluidic network through which the microfluidic samples flow.For example, a plurality of gas actuators can be fabricated in the samesubstrate which forms the microfluidic network. One such gas actuator iscoupled to the network at a first location for providing gas pressure tomove a microfluidic sample within the network. Another gas actuator iscoupled to the network at a second location for providing gas pressureto further move at least a portion of the microfluidic sample within thenetwork.

[0010] In other aspect, the invention relates to the use of valves withthe plurality of actuators. For example, in one embodiment, a valve iscoupled to a microfluidic network so that, when the valve is closed, itsubstantially isolates the second gas actuator from the first gasactuator. Such valves can control the direction of the propulsive forceof the actuatators by preventing the expanding gas from traveling incertain directions, while permitting it to expand in the desireddirection. They also extend the range over which an actuator can propela microdroplet, by preventing the gas from dissipating in certain inareas upstream from the microdroplet.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The present invention is described below in reference to thefollowing drawings, in which:

[0012]FIG. 1 shows a microfluidic system according to the invention;

[0013]FIG. 2 shows an expanded view of a microfluidic device.

[0014]FIG. 3 shows a schematic of a microfluidic device of themicrofluidic system of FIG. 1;

[0015]FIG. 4, shows a top view of the microfluidic device of FIG. 3;

[0016]FIG. 5 shows a partial cross-sectional view of the microfluidicdevice of FIG. 4;

[0017]FIG. 6 shows a partial cross-sectional view of an upper substratefrom the microfluidic device of FIG. 2;

[0018]FIG. 7 shows a second partial cross-sectional view of an uppersubstrate from the microfluidic device of FIG. 2;

[0019]FIG. 8a shows a top view of a microdroplet preparation zone of themicrofluidic device of FIG. 4 before preparation of a microdroplet;

[0020]FIG. 8b shows cross sectional view of the microdroplet preparationzone of FIG. 8a;

[0021]FIG. 9a shows a top view of a microdroplet preparation zone of themicrofluidic device of FIG. 4 after preparation of a microdroplet;

[0022]FIG. 9b shows a cross sectional side view of the microdropletpreparation zone of FIG. 9a;

[0023]FIGS. 10a-10 c show cross sectional side views of a capillaryassisted fluid barrier of the present invention;

[0024]FIGS. 11a-11 c show top views of a fluid barrier comprising avent;

[0025]FIGS. 12a and 12 b show top views of the lysing module of themicrofluidic device of FIG. 4, before and after preparation of a lysedsample;

[0026]FIGS. 13a and 13 b show a second embodiment of a lysing module ofthe invention;

[0027]FIG. 14 shows a pulsing circuit associated with the lysing moduleof FIG. 4; and

[0028]FIGS. 15a-15 c show a second microdroplet preparation module ofthe invention.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT

[0029] The present invention relates to microfluidic systems and methodsfor processing materials, such as samples and reagents. Morespecifically, the invention relates to microfluidic systems and methodsfor moving fluids within a microfluidic system. In the embodimentdescribed below, the fluid includes particles which tend to move withthe fluid. The fluid component of the particle-containing fluid is a gasor, preferably, a liquid. The particles of the particle-containing fluidare preferably whole cells, such as bacterial cells or cells of ananimal, such as a human. However, they may include intracellularmaterial from such cells. For example, a system of the invention may beused to process a sample of bacterial cells to determine whether thebacteria are pathogenic.

[0030] A. System Overview

[0031]FIG. 1 depicts a microfluidic system 100 that includes amicrofluidic device 110 and corresponding cartridge 120, which receiveone or more fluid samples and process the samples under the control ofcomputer 127 and data acquisition and control board (DAQ) 126.

[0032] Computer 127 preferably performs high level functions, such assupplying a user interface that allows a user to select desiredoperations, notifying the DAQ 126 as to the selected operations, anddisplaying for the user the results of such operations. These operationsinclude, for example, subjecting a sample to process steps within thevarious process zones of the microfluidic device. The computer 127 maybe a portable computer to facilitate transport of the microfluidicsystem.

[0033] Computer 127 is connected to DAQ 126 via connection 128, whichprovides data I/O, power, ground, reset, and other functionalconnectivity. Alternatively, a wireless link 132 between the computer127 and the DAQ 126 may be provided for data and control signal exchangevia wireless elements 132(a) and 132(b). Where the data link is awireless link, for example, the DAQ 126 may have separate power source,such as a battery.

[0034] In general, DAQ 126 controls the operation of microfluidic device110 in accordance with the high level instructions received fromcomputer 127. More specifically, to implement a desired operationrequested by computer 127, DAQ 126 supplies the appropriate electricalcontrol signals to cartridge 120 via contacts 125.

[0035] Cartridge 120 provides electrical and optical connections 121 forelectrical and optical signals between the DAQ 126 and the microfluidicsubstrate 110, thereby allowing DAQ 126 to control the operation of thesubstrate.

[0036] The chip carrier cartridge 120 is shown being inserted into (orremoved from) an interface hardware receptacle of the DAQ 126 havingelectrical and optical contacts 125 standardized to mate with acorresponding contacts 121 of the chip carrier cartridge 120. Mostcontacts are for electrical signals, while certain ones are for opticalsignals (IR, visible, UV, etc.) in the case of optically-monitored oroptically-excited microfluidic processors. Alternatively (not shown),the entire DAQ 126 may be a single ASIC chip that is incorporated intothe Chip Carrier Cartridge 120, wherein contacts 121,125 would becomeconductive pathways on a printed circuit board.

[0037] B. Microfluidic Device

[0038]FIG. 2 illustrates the general structure of a preferred type ofmicrofluidic device. The device includes an upper substrate 130, whichis bonded to a lower substrate 132 to form a fluid network.

[0039] The upper substrate 130 depicted in FIG. 2 is preferably formedof glass and has a microfluidic network 134 in its bottom surface 136.Those skilled in the art will recognize that substrates composed ofsilicon, glass, ceramic, plastic, and/or quartz are all acceptable inthe context of the present invention.

[0040] The microfluidic network includes a plurality of zones. Thenumber of zones, as well as the overall topology of the microfluidicnetwork, will depend upon the particular application which themicrofluidic device is designed to perform. The zones of themicrofluidic device may have any cross-sectional shape, such asgenerally arcuate or generally polygonal. For example, a zone mayinclude channels, chambers or other substantially enclosed spaces. By“substantially enclosed” it is meant that materials enter or exit thezones only through predetermined pathways. Examples of such pathwaysinclude channels, microchannels and the like, which interconnect thevarious zones. The zones preferably have at least one micro-scaledimension, such as less than about 250 μm or, more preferably, less thanabout 75 μm.

[0041] The channels and chambers of the microfluidic network are etchedin the bottom surface 136 of the upper substrate 130 using knownphotolithographic techniques. More specifically, transparent templatesor masks containing opaque designs are used to photo-define objects onthe surface of the substrate. The patterns on the templates aregenerated with computer-aided-design programs and can delineatestructures with line-widths of less than one micron. Once a template isgenerated, it can be used almost indefinitely to produce identicalreplicate structures. Consequently, even extremely complex microfluidicnetworks can be reproduced in mass quantities and at low incrementalunit cost. Alternatively, if a plastic material is used, the uppersubstrate may be formed using injection molding techniques, wherein themicro-channels are formed during the molding process.

[0042] The lower substrate 132 may include a glass base 138 and an oxidelayer 140. Within oxide layer 140, resistive heaters 142 and electricleads 144 are formed using photolithographic techniques. The leads 144connect to terminals 146 which are exposed at the edge of the substrateto permit electrical connection to cartridge 120, thereby permittingDAQ126 to control the heaters. More specifically, to activate a heater142, DAQ 126 applies a voltage across a pair of terminals 146 (viacartridge 120) to supply current through leads 146 and heater 142,thereby heating the resistive heater element 142.

[0043] Metal heater elements 142 are positioned so that, when the upperand lower substrates are bonded together, the heaters reside directlybeneath certain regions of the fluid network of the upper substrate soas to be able to heat the contents of these regions. The silicon oxidelayer 140 prevents the heating elements 142 from directly contactingwith material in the microfluidic network.

[0044] The oxide layer 140, heating elements 142, and resistive leads144 are fabricated using well-known photolithographic techniques, suchas those used to etch microfluidic network.

[0045]FIG. 3 illustrates a top-down view of microfluidic device 110. Asshown, the substrate has a sample input module 150 and reagent inputmodule 152 to allow sample and reagent materials, respectively, to beinput to device 110. Preferably, input modules 150, 152 are disposed toallow automatic material input using a computer controlled laboratoryrobot 154.

[0046] The substrate also includes process modules 156, 158, 160, 166and 162 for processing the sample and reagent materials. Within theseprocess modules, a sample may be subjected to various physical andchemical process steps. For example, enrichment module 156 prepares afluid sample having a relatively high concentration of cell particles,lysing module 160 releases intracellular material from the cellparticles, and mixing module 166 mixes the resultant sample with certainreagents. As another example, an amplification process module 162 may beused to amplify and detect minute quantities of DNA within a sample.

[0047] Various modules of microfluidic device 110 are connected, such asby channels 164, to allow materials to be moved from one location toanother within the device 110. Actuators 168, 170, 172 associated withthe microfluidic device provide a motive force, such as a gas pressure,to move the sample and reagent material along the channels and zones.For example, a first actuator 168 moves material downstream from processmodule 156 to process module 158. Upon completion of processing withinprocess module 158, a second actuator 170 moves material downstream tomixing process module 160. Subsequently, actuator 170 or an additionalactuator moves the material to mixing module 166, where the materialmixes with a reagent moved by actuator 172. Finally, actuator 172, oranother actuator, moves the mixed material to module 162.

[0048] Because each actuator is preferably responsible for movingmaterials within only a subset of the modules of device 110, samplematerials can be controlled more precisely than if a single actuatorwere responsible for moving material throughout the entire device. Thevarious functional elements, of microfluidic device 110, including theactuators, are preferably under computer control to allow automaticsample processing and analysis.

[0049] C. Multiple Actuators

[0050] The various actuators of microfluidic device 110 cooperate tomove material between different locations of microfluidic device 110.For example, actuator 168 moves material, such as an enriched sample,between an enrichment zone 931 and a microdroplet preparation module158. Actuator 170 prepares a microdroplet from the enriched sample and,in so doing, moves the microdroplet to a lysing zone 950. Actuator 170is used to move material from the lysing zone 950 to mixing module 166.It should be noted, however, that another actuator may be disposedintermediate between lysing zone 950 and microdroplet preparation zoneto move the lysed sample downstream to the mixing module 166.

[0051] Actuators of device 110 may also cooperate in moving two amountsof material simultaneously. For example, as described above, actuator172 and actuator 170 cooperate to mix reagent and lysed microdroplets.Such cooperative actuators can be controlled independently of oneanother to ensure proper mixing. For example, if one material is knownto be more viscous, the motive force moving that material can beincreased independently of the motive force moving the other material.

[0052] The multiple actuators and modules of microfluidic device 110 arepreferably operatively connectable and isolatable by the valves ofmicrofluidic device. For example, a closed state of either of valves915, 216 operatively isolates microdroplet preparation module 170 fromenrichment module 156. Thus, one or more actuators can be used to movematerials between predetermined locations within microfluidic device110, without perturbing or contacting material present in an operativelyisolated module. The ability to operatively connect and isolate desiredmodules is advantageous in microfluidic devices having many processfunctions. Further, these valves also control the direction of thepropulsive force of the actuatators by preventing the expanding gas fromtraveling in certain directions, while permitting it to expand in thedesired direction. This also extends the range over which an actuatorcan propel a microdroplet, by preventing the gas from dissipating incertain in areas upstream from the microdroplet.

[0053] The following demonstrates the cooperative operation of suchmultiple actuators in an example embodiment having a plurality ofprocessing modules, namely an enrichment zone 915, a microdropletpreparation module 158, a cell lysing module 160, a mixing module 166and a DNA manipulation module 167.

[0054] 1. Enrichment Module

[0055] a. Structure of Enrichment Module

[0056] Referring to FIGS. 4 and 5, a microfluidic device 901 includes anenrichment module 156 for concentrating samples received therein. Thesesamples include particle-containing fluids, such as bacterialcell-containing fluids. In general, enrichment module 156 receives aflow of particle-containing fluid from an input port 180 of input module150, and allows the fluid to pass through the zone while accumulatingparticles within the zone. Thus, as more fluid flows through the zone,the particle concentration increases within the module. The resultantconcentrated fluid sample is referred to herein as an enriched particlesample.

[0057] The enrichment module includes an enrichment zone 931 (FIG. 5), aflow through member 900, valves 915, 919, and sample introductionchannel 929. Valve 919 is connected between the flow through member 900and actuator 168 as shown, and valve 915 is connected between the flowthrough member and a down stream channel 937 which leads to processmodule 158. These valves may be of any type suitable for use in amicrofluidic device, such as thermally actuated valves, as discussed inco-pending application Ser. No. 09/953,921, filed Sep. 9, 2001. Thevalves may be reversible between the open and closed states to allowreuse of enrichment module 931.

[0058] The flow through member is also connected to the sample inputmodule 150 via the sample introduction channel 929 to allow fluid toflow into the enrichment zone. Valve 913 is connected to this sampleintroduction channel to control the in-flow and outflow of fluid fromthe input port.

[0059]FIG. 5 is a cross-sectional view of the enrichment zone whichshows the flow through member in greater detail. As shown, flow throughmember 900 has first and second surfaces 941, 943. First surface 941 ispreferably adjacent enrichment chamber 931. Second surface 941 ispreferably spaced apart from the enrichment chamber 931 by flow throughmember 900. Flow through member 900 is preferably formed of a materialhaving pathways smaller than the diameter of the particles to beenriched, such as pores of less than about 2 microns in diameter, forexample, about 0.45 microns. Suitable materials for constructing flowthrough member 900 include, for example, filter media such as paper ortextiles, polymers having a network of pathways, and glassy materials,such as glass frits.

[0060]FIGS. 6 and 7 depict cross sectional views of upper substrate 130that illustrate an enrichment zone 931. As shown, fluid exits enrichmentzone 931 through surface 941, passes through member 900 and enters aspace 400. Space 400 may include an absorbent material 402 to absorb theexiting fluid. Thus, space 400 preferably provides a substantiallyself-contained region in which fluid exiting the enrichment zone cancollect without contacting exterior portions of the microfluidic system100.

[0061] Space 400 is formed during the fabrication of upper substrate130. As discussed above, microfluidic features, such as zones andchannels, are fabricated at surface 136 of substrate 130. Space 400,however, is fabricated at a surface 137, which is preferably disposed onthe other side of substrate 130, opposite surface 136. Thus, even whensurface 136 is mated with lower substrate 132, fluid can exit enrichmentzone 931 via flow through member 900.

[0062] Flow through member 900 and absorbent material 402 do not requireadhesives or other fasteners for positioning within substrate 130.Rather flow through member 900 and absorbent material 402 may be formedof a shape and size that substantially corresponds to space 400.Friction then holds flow through member 900 and absorbent material 402in place once they are positioned in space 400. Any residual gap atlocations 404 between flow through member 900 and substrate 130 shouldbe small enough to prevent particles from exiting enrichment zone 931through the gap 404. Naturally, adhesive or other fastening means may beused to secure flow through member 900 or absorbent material 402.

[0063] In an alternative embodiment, a flow through member is formedintegrally with a substrate by using microfabrication techniques, suchas chemical etching, that introduce pores or other pathways into thesubstrate. The pores provide fluid passage between enrichment zone 931and an outer portion of the substrate.

[0064] b. Operation of Enrichment Module

[0065] To enrich a sample, the device 901 operates as follows.Referrring to FIG. 4, valves 915, 919 are initially closed, and valve913 is open. A particle-containing fluid is introduced into input port180. Since valve 913 is open, it allows the sample to pass along channel929 into enrichment zone 931. Alternatively, enrichment zone 931 can beconfigured to receive samples directly, such as by injection. Sincevalves 915 and 919 are closed, fluid is substantially prevented fromescaping into actuator 977 and downstream channel 937.

[0066] Thus, flow through member 900 provides the only path for fluid toexit the enrichment channel. Fluid passes through surface 941 and exitsenrichment zone 931 via second surface 943, while particles accumulatewithin the zone. Enrichment zone 931 can therefore receive a volume offluid that is larger than the volume of the enrichment chamber 931.Thus, as fluid flows through the chamber, the concentration of particleswithin the chamber increases relative to the concentration in theparticle-containing fluid supplied at the sample input. Where theparticles are cells, the concentration or number of cells in zone 931preferably becomes great enough to perform a polymerase chain reaction(PCR) analysis of polynucleotides released from the cells in adownstream processing module.

[0067] Enrichment zone 931 thus prepares an enriched particle samplefrom particles of particle-containing fluids received therein. Theenriched particle sample has a substantially higher ratio of particlesper volume of fluid (PPVF) than the corresponding ratio of theparticle-containing fluid received by the enrichment zone. The PPVF ofthe enriched particle sample is preferably at least about 25 times,preferably about 250 times, more preferably about 1,000 times greaterthan the PPVF of the particle-containing fluid.

[0068] After a sufficient volume of particle containing fluid has beenreceived by enrichment zone 931, valve 913 is closed thereby blockingfurther flow of fluid into the enrichment zone, and preventing materialin zone 931 from returning to the sample introduction port 180. Valves915, 919 are then opened, preferably upon actuating heat sourcesassociated therewith. When opened, valve 919 allows actuator 168 to pushenriched sample, and valve 915 allows the enriched sample to movedownstream.

[0069] Actuator 168 provides a motive force that moves the enrichedparticle sample from enrichment zone 931. Actuator 168 is preferably agas actuator, which provides a gas pressure upon actuation of a heatsource 975, which is in thermal communication with a volume of gas 977.Actuation of heat source 975 raises the temperature and, therefore thepressure, of gas 977. The flow through member and the fluid thereinsubstantially prevents gas from escaping the enrichment zone. Thus, theresulting gas pressure moves the enriched particle sample downstreamfrom the enrichment zone 931.

[0070] The gas actuator may include elements to facilitate alternativepressure generation techniques such as chemical pressure generation. Inanother embodiment, the actuator may decrease a volume of gas associatedwith an upstream portion of the enrichment zone to thereby create apressure differential across the sample that moves the sample from theenrichment zone. An example of such an element is a mechanical actuator,such as a plunger or diagram.

[0071] Rather than generating a positive pressure upstream from theenrichment zone, the gas actuator may decrease a pressure downstreamfrom the zone relative to a pressure upstream. For example, the gasactuator may include a cooling element in thermal contact with a volumeof gas associated with a downstream portion of the zone. Contraction ofthe gas upon actuating the cooling element creates a gas pressuredifference between the upstream and downstream portions of theenrichment zone to move the enriched particle sample from the enrichmentzone. Alternatively, a mechanical actuator may be used increase a volumeof gas associated with a downstream portion of the enrichment zone tothereby decrease the pressure of the gas and move the enriched particlesample from the enrichment zone.

[0072] The enriched particle sample is preferably moved downstream withessentially no dilution thereof, i.e., the concentration of the enrichedparticles is not substantially decreased upon movement from theenrichment zone 931. Thus, removal of particles from the enrichmentchannel of the present invention does not require diluting or otherwisecontacting the particles with a fluid different from the fluid of theparticle-containing fluid introduced to the enrichment channel. Incontrast, in systems that concentrate substances by surface adsorption,removal of the adsorbed substances requires an elution fluid, whichcontacts and thereby dilutes the substances.

[0073] Upon removal from the enrichment zone of the present invention,the enriched particle sample is preferably received by downstreamchannel 937. Downstream channel 937 leads to other processing modules,which perform further processing of the enriched particle sample. In theembodiment of FIG. 3, the enriched particle sample is received by amicrodroplet preparation module 158, which prepares a microdropletsample comprising a portion of the enriched particle sample.

[0074] 2. Microdroplet Preparation Module

[0075] a. Characteristics of a Microdroplet

[0076] A microdroplet 802 is a discrete sample having a predeterminedvolume between, for example, about 1.0 picoliter and about 0.5microliters. Thus, microdroplets prepared by microdroplet preparationmodule provide a known amount of sample for further processing. Thevolume of the microdroplet prepared by the microdroplet preparationmodule is preferably essentially independent of the viscosity,electrical conductivity, and osmotic strength of the fluid of themicrodroplet.

[0077] Microdroplet 802 is preferably defined by upstream and downstreamboundaries each formed by a respective gas liquid interface 804, 806.The liquid of the interface is formed by a surface of a liquid formingthe microdroplet. The gas of the interface is gas present in thechannels microfluidic of microfluidic device 901.

[0078] b. Structure and Operation of the Microdroplet Preparation Module

[0079] Referring to FIGS. 8a-8 b and 9 a-9 b, microdroplet preparationmodule 158 prepares a microdroplet 802 from a microfluidic samplereceived therein. This module includes a microdroplet preparation zone800, a positioning element 979, a gas actuator 170, and a valve 216which cooperate to prepare microdroplet 800 from microfluidic samplesreceived from the enrichment zone.

[0080] As explained above, actuator 168 of the enriched zone pushes theenriched sample into the microdroplet preparation zone 800. The enrichedsample moves until reaching positioning element 979. In general, apositioning element inhibits the downstream progress of a microfluidicsample to thereby position the sample at a desired location. However, asexplained more fully below, the positioning element does not permanentlyinhibit progress of the sample. Rather, it allows the microfluidicsample to continue downstream at a predetermined later time.

[0081] The leading edge of microfluidic sample 808 that reachespositioning element 979 is positioned downstream from an opening 820 ofgas actuator 170. Accordingly, a first portion 821 of microfluidicsample 808 is disposed upstream from opening 820 and a second portion822 of microfluidic sample 808 is disposed downstream from opening 820.

[0082] Referring to FIGS. 8a-8 b, gas actuator 170 is actuated, such asby DAQ 126, to thereby generate a gas pressure sufficient to separatemicrodroplet 802 from the second portion 822 of microfluidic sample 808.The gas pressure is preferably provided by the actuation of a heatsource 958, which heats a volume of gas associated with gas actuator957. As the pressure increases, the gas expands, thereby separating amicrodroplet 802 from the rest of sample 808. Microdroplet 802 maycomprise only a portion, such as less than about 75%, or less than about50%, of microfluidic sample 808 received by microdroplet preparationzone 800. The dimensions of microdroplet 802 are determined by thevolume of the channel between fluid barrier 979 and opening 820. Forexample, for a channel having a uniform cross-sectional area, a lengthl₁ of microdroplet 802 corresponds to a distance d₄ between positioningelement 979 and opening 820. Thus, a microfluidic device can beconfigured to prepare microdroplets of any volume by varying the lengthbetween the fluid barrier and corresponding actuator opening.

[0083] Continued actuation of gas actuator 170 overcomes the inhibitoryeffect of positioning element 979, thereby driving microdroplet 802 to alocation downstream of microdroplet preparation zone 800 while thesecond portion 822 of the microfluidics sample moves upstream frommicrodroplet 802 to cell lysis module 160.

[0084] 3. Cell Lysis Module

[0085] Referring back to FIG. 3, a lysing module 160 receives themicrodroplet 802 prepared by microdroplet preparation zone 800. Ingeneral, lysing module 160 releases material from inside the particles,such as by releasing intracellular material from cells.

[0086] As shown in FIGS. 4 and 12, lysing module 160 includes a lysingzone 950, a lysing mechanism within the lysing zone (such as electrodes954), and a vented positioning element 200 positioned upstream from thelysing zone. The lysing mechanism preferably includes a set ofelectrodes or other structures for generating electric fields within thelysing zone. The vented positioning element preferably includes a vent202, a valve 204, and a second positioning element 206 for inhibitingfluid from flowing into the vent.

[0087] As explained above, actuator 170 of the microdroplet preparationmodule 158 drives a microdroplet into cell lysis module 160. As themicrodroplet moves into module 160, vented positioning element 200positions microdroplet 802 in a lysing position with respect toelectrodes 954. More specifically, as the microdroplet arrives in lysingmodule 160 it passes the opening of positioning element 200, becausesecond positioning element 206 inhibits the microdroplet from flowinginto vent 202. When the rear end of the microdroplet passes the openingof barrier 200, the propulsion gas from actuator 170 dissipates throughvent 202, thereby substantially equalizing gas pressure upstream ofmicrodroplet 802 with a pressure downstream of microdroplet 802. Thus,the microdroplet stops movement at a lysing position just downstreamfrom barrier 200. Preferably, in the lysing position, substantially allof microdroplet 802 is disposed between an upstream edge 212 and adownstream edge 214 of electrodes 954.

[0088] After microdroplet 802 is placed in the cell lysing position, apulse circuit of DAQ 126 supplies a pulsed voltage signal acrosselectrodes 954. In response, electrodes 954 generate a pulsed electricfield in the vicinity of the electrodes. Because the microdroplet isposition in this vicinity, cells within the microdroplet are subjectedto the pulsed field. Preferably, substantially all of the cells, such asgreater than about 75%, of the microdroplet are subjected to an electricfield sufficient to release intracellular material therefrom. The lysingmodule thus prepares a lysed microdroplet comprising a predeterminedamount of sample.

[0089] A preferred pulse circuit is shown in FIG. 14. In general, thiscircuit generates a sequence of voltage pulses that yields acorresponding sequence of electrical field pulses in the vicinity ofelectrodes 954 having an amplitude and duration sufficient to release adesired amount of intracellular material from cells within themicrodroplet.

[0090] Intracellular material present in lysed microdroplet isaccessible to further process steps. For example, DNA and/or RNAreleased from cells is accessible for amplification by a polymerasechain reaction. As used herein, the term lysing does not require thatthe cells be completely ruptured. Rather, lysing refers to the releaseof intracellular material. For example, rather than rupturing the cells,the electric field may increase the porosity of cell membranes by anamount that allows release of intracellular material without permanentrupture of the membranes.

[0091] Other lysing mechanisms may also be employed to releaseintracellular material from cells. For example, material may be releasedby subjecting cells to other forces including for example osmotic shockor pressure. Chemicals, selected from the group of surfactants,solvents, and antibiotics may be contacted with the cells. Mechanicalshear methods may also be used to release intracellular materials.

[0092] The lysed microdroplet may be moved downstream to mixing module160 for further processing. To move lysed microdroplet downstream, valve216, which is disposed upstream of lysing zone 950, is closed. Valve 204is also closed to prevent gas from exiting lysing zone 950 via vent.Actuator 170 is then actuated, as described above, to provide a gaspressure sufficient to move lysed microdroplet downstream of lysing zone950.

[0093] In an alternative embodiment, a lysing module 300, as shown inFIGS. 13a, 13 b, includes a lysing zone 302 which is configured toprepare a lysed microdroplet 304 of predetermined volume from amicrofluidic sample 306, which may have an indeterminate volume. Lysingzone 302 preferably includes a lysing mechanism such as electrodes 308.Electrical leads 310 provide a connection to a pulse circuit of DAQ 126,via contacts 112, chip carrier 120, and contacts 125. A positioningelement 312 is disposed downstream of lysing zone 302. An actuator 314is disposed upstream from lysing zone. Actuator 314 preferably includesa second positioning element 316 to prevent fluid from the microfluidicsample from entering therein.

[0094] Lysing zone 302 operates as follows. The microfluidic sample 306enters lysing zone 302 and moves downstream until a downstream interface316 of the microfluidic sample 306 encounters positioning element 312.The positioning element 312 preferably increases a surface tension ofthe downstream interface of the microfluidic sample 306, therebyinhibiting further downstream movement and positioning a portion of themicrofluidic sample in a lysing position with respect to electrodes 308.The lysing position is defined as the location of the portion of themicrofluidic sample disposed downstream of actuator 314 and upstream ofpositioning element 312. Preferably, actuator 314 and positioningelement 312 are disposed adjacent electrodes 308 such that substantiallyall of the material present in the lysing position is subjected to theelectric field upon actuating electrodes 308.

[0095] Actuation of electrodes 308 in the embodiment described above,provides an electrical field sufficient to release intracellularmaterial from cells present in the portion of the microfluidic sample inthe lysing position. Once a sufficient amount of intracellular materialhas been released, actuator 314 is actuated to prepare lysedmicrodroplet 304 from the microfluidic sample 306. Actuator 314preferably provides a gas pressure sufficient to move the lysedmicrodroplet 304 to a downstream portion of a microfluidic device suchas mixing module 166.

[0096] 4. Mixing Module and Reagent Input Module

[0097] Referring back to FIG. 4, a lysed sample prepared by lysingmodule 160 is received by mixing module 166. Mixing module 166 includesa mixing zone 958. In this zone, the lysed cell sample is contacted,such as by mixing, with an amount of reagent received from the reagentsource module 152. Reagent source module 152 includes a reagentmicrodroplet preparation zone (RMPZ) 434, which preferably operates toprepare a microdroplet having a predetermined volume of reagent.

[0098] a. Reagent Input Module

[0099] Reagent input module 152 is essentially the same as microdropletformation module 158, however, it is specifically designed for formationof a microdroplet of reagent having a predetermined volume which willyield a desired ratio of reagent to sample when mixed with themicrodroplet from cell lysing module 160. Module 152 includes an inputport 420, a valve 422, and an actuator 172, each of which joins areagent source channel 428. An overflow channel 424, which also joinsreagents source channel 428, may also be provided. Actuator 172 mayinclude a second positioning element 432 to prevent liquid from enteringtherein.

[0100] Reagent materials, which preferably comprise at least one liquid,are introduced via input port 420, such as with a pipette or syringe.Examples of suitable reagent materials include substances to facilitatefurther processing of the lysed cell sample, such as enzymes and othermaterials for amplifying DNA therein by polymerase chain reaction (PCR).The reagent material moves downstream within reagent source channel 428until a downstream portion of the reagent material contacts apositioning element 426. Any additional reagent material that continuesto be received within reagent source module preferably enters overflowchannel 424. When the introduction of reagent is complete, valve 422 isclosed to prevent reagent from exiting reagent source channel viareagent source port 420.

[0101] b. Mixing Module

[0102] Mixing zone 958 of the mixing module includes adjoined first andsecond channels 410, 412. Materials moving downstream toward mixing zone958 contact one another and preferably mix therein. Because of themicro-scale dimensions of mixing zone 958, the sample and reagentmaterials preferably mix by diffusion even in the absence of othersources of mass transport, such as mechanical agitation. It should beunderstood however, that agitation forces, such as acoustic waves may beapplied to enhance mixing within mixing zone 958.

[0103] c. Operation of Mixing Module and Reagent Input Module

[0104] Reagent source module 152 and mixing module 166 preferablyoperate as follows. When a lysed sample from lysing zone 950 is ready tobe mixed with reagent material, actuator 172 is actuated to prepare amicrodroplet of reagent. The microdroplet of reagent is prepared fromthe portion of reagent material downstream of an opening 430 of actuator172 and upstream of positioning element 427. Thus, assuming that thedimensions of the reagent source channel 428 are constant, the volume ofthe microdroplet of reagent is determined by the distance between thepositioning element 426 and the actuator opening 430.

[0105] The microdroplet of reagent moves downstream toward channel 412of reagent mixing zone. Meanwhile, a sample of lysed material, such as alysed microdroplet, is moved downstream from lysing zone 950 towardchannel 410 of mixing zone 958. Actuator 170 may provide the motiveforce to move the lysed microdroplet downstream. Alternatively, asdiscussed above, another actuator may be disposed upstream of lysingzone 950 but downstream of actuator 170 to provide the necessary motiveforce.

[0106] The sample and reagent material enter a downstream channel 438 ofmixing zone 958, where the materials contact and mix. Because both thelysed sample and reagent material are mixed in the form ofmicrodroplets, mixing zone 958 prepares an amount of mixed materialhaving a predetermined ratio of sample to reagent. The volumes ofmicrodroplets prepared within microfluidic device 110 are preferablyindependent of physical properties, such as viscosity, electricalconductivity, and osmotic strength, of the microdroplets. Thus, mixingzone 958 prepares an amount of mixed material having a sample to reagentmaterial that is also independent of the physical and chemicalproperties of the mixed materials. A vent 440, which is downstream ofthe various zones of the microfluidic device 110 ensures that downstreampressure buildup does not inhibit downstream movement of samples withinmicrofluidic device 110.

[0107] 5. DNA Manipulation Module

[0108] The mixed lysed cell sample and reagent are received within a DNAmanipulation zone 971 of DNA manipulation module 162. Module 162 canperform, for example, restriction, digestion, ligation, hybridizationand amplification of DNA material. In one embodiment, DNA manipulationzone 971 is configured to perform PCR amplification of nucleic acidspresent within the lysed cell sample. Vent 440 prevents pressure fromincreasing within zone 971 as the lysed cell sample and reagent arebeing introduced thereto. Valves 972 and 973 of DNA manipulation module162 may be closed to prevent substances therein zone from exiting, suchas by evaporation, during PCR amplification. The DNA manipulation zoneis configured with heat sources under control of computer 127 to allowthermal cycling of DNA manipulation zone during amplification, asunderstood by one of skill in the art.

[0109] System 901 includes also includes a detector 981 to detect thepresence of amplified polynucleotides produced by PCR. Detector 981 ispreferably an optical detector in optical communication, such as by afiber optic 981, with zone 971. A light source, such as a laser diode,introduces light to DNA Manipulation zone 971 to generate fluorescenceindicative of the amount of amplified polynucleotides present therein.The fluorescence arises from fluorescent tags, included in the reagentand associated with the polynucleotides upon amplification.

[0110] C. Preferred Positioning Elements

[0111] Preferred positioning elements are discussed below.

[0112] 1. Non-Wetting Positioning Elements

[0113] A positioning element 979 may be formed by a non-wetting materialdisposed to contact a microfluidic sample. The physio-chemicalproperties of the non-wetting material are chosen upon considering thetype of liquid forming the microfluidic sample. For example, where themicrofluidic sample is an aqueous sample, the positioning elementpreferably comprises a hydrophobic material. An exemplary hydrophobicmaterial includes a non-polar organic compound, such as an aliphaticsilane, which can be formed by modifying an internal surface ofmicrofluidic device 901. For microfluidic samples formed of organicsolvents, the non-wetting material may comprise a hydrophilic material.

[0114] When microfluidic sample 808 encounters positioning element 979,the liquid of the microfluidic sample experiences an increased surfacetension at downstream interface 810, which increased surface tensioninhibits continued downstream motion of microfluidic sample 808.Increasing the gas pressure difference between upstream and downstreamportions of the microfluidic sample overcomes the resistance and movesthe microfluidic sample downstream.

[0115] 2. Capillary Assisted Positioning Elements

[0116] Referring to FIGS. 10a-10 c, another type of positioning elementmay be formed by modifying the dimensions of the microfluidic channel toform a capillary assisted positioning element (CAFB) 700. A CAFBcomprises an upstream feed zone 702, a loading zone 704, and a stop zone704. A microfluidic sample 720 encountering the CAFB moves downstreamuntil a downstream interface 710 of the microfluidic sample contactsupstream surfaces 714 of the loading zone 706. At this point, capillaryaction causes the microfluidic sample to move downstream until thedownstream sample interface 710 encounters the opening 712 between theloading zone 704 and the stop zone 706. Surface tension resists thetendency of the microfluidic sample to continue downstream past opening714. Thus, the microfluidic sample 720 is positioned at a predeterminedlocation along the channel axis with respect to positioning element 700.

[0117] The volume of the microfluidic sample encountering the CAFBpreferably has a larger volume than a volume of the loading zone 704 toensure that the microfluidic sample will advance fully to opening. Forfluids that have similar surface tensions and interface properties aswater, the depth d₁ of the loading zone 704 is preferably about 50% orless of the respective depths d₂, d₃ of the stop and feed zones.

[0118] The tendency of a microfluidic sample to move in a givendirection is governed by the ratio between the mean radius of curvature(MRC) of the front of the microfluidic sample and the MRC of the back ofthe microfluidic sample. These curvatures depend upon the contact angleof the fluid of the sample and the dimensions of the zone in which themicrodroplet is moving. A MRC r₁ of a microdroplet interface in theloading zone is preferably smaller than a MRC r₂ of a droplet interfacewithin the feed zone or a MRC r₃ of a droplet interface within the stopzone. The MRC r₂ is preferably larger than the MRC r₃. Thus, the radiusof curvature of the downstream microdroplet interface increases uponencountering the stop zone thereby inhibiting further downstreammovement. Preferably, the contact angle of the fluid with the wall issubstantially constant throughout the capillary assisted loading zone.

[0119] 3. Vented Positioning Elements

[0120] Referring to FIGS. 11a-11 c, a positioning element 500 operatesto position a microfluidic sample 502 by reducing the gas pressureacting upon an upstream portion 504 of the microfluidic sample relativeto the gas pressure acting upon a downstream portion 506 of themicrofluidic sample. Positioning element 500 includes a vent 508disposed in gaseous communication with a zone 510 along whichmicrofluidic sample 502 moves. Vent 508 preferably communicates withzone 510 via a passage 526. The zone may be for example, a channel orconduit. Positioning element 500 may also include a second positioningelement 516, such as a non-wetting material, to substantially preventfluid from the microfluidic sample from contacting the vent.

[0121] An open state of a valve 512 allows passage of gas between zone510 and vent 508. A closed state of valve 512 prevents such passage ofgas. Valve 514 is preferably thermally actuated and includes a mass 514of TRS.

[0122] An actuator 518 is disposed upstream of positioning element 500.Actuator 518 is preferably a gas actuator and may include a heat source520 to heat a gas associated with actuator 518. Actuator 518 may includea positioning element 522, such as non-wetting material, tosubstantially prevent fluid from the microfluidic sample from enteringtherein.

[0123] Positioning element 500 preferably operates as follows. Referringto FIG. 11a, microfluidic sample 502 moves downstream in the directionof arrow 524. Microfluidic sample is preferably moved by a gas pressureprovided from an upstream actuator, which is not shown in FIGS. 9a-9 c.The gas pressure acts upon upstream portion 504.

[0124] Referring to FIG. 11b, when upstream portion 504 passes theopening of vent 508, the upstream gas dissipates through vent 508,thereby reducing the upstream pressure. The pressure reduction, whichpreferably equalizes the downstream and upstream pressures, reduces oreliminates the motive force tending to urge the microfluidic sampledownstream.

[0125] Referring to FIG. 11c, valve 512 is closed to prevent passage ofgas between zone 510 and vent 508. Preferably, TRS 514 moves intopassage 526. Upon closing valve 512, the actuation of actuator 518provides a motive force to move microfluidic sample 502 downstream inthe direction of arrow 528 for further processing.

[0126] 4. Active Fluid Positioning Elements

[0127] Referring to FIGS. 15a-15 c, a microdroplet preparation module652 has a microdroplet preparation zone 650, an active fluid positioningelement 654, an actuator 656, and a valve 658. A second actuator 660 isoperatively associated with the active positioning element 654 tointroduce a microfluidic sample 666 to the microdroplet preparation zone650. Second actuator 660 is preferably located upstream from valve 658.Microdroplet preparation module 652 prepares a microdroplet 668, whichhas a predetermined volume from the microfluidic sample 666 receivedtherein.

[0128] In operation, microfluidic preparation module 652 receives themicrofluidic sample 666, which moves downstream because of a motiveforce provided by the second actuator 660. The motive force ispreferably an upstream gas pressure, which is greater than a downstreamgas pressure acting upon the microfluidic sample 666. The microfluidicsample moves downstream until a downstream portion 670 thereofencounters active positioning element 654, which preferably comprises asensor 672 having electrical leads 674. The leads 674 are in electricalcommunication with I/O pins of the microfluidic device to allow signalsfrom sensor 672 to be received by a DAQ.

[0129] Sensing element 672 is preferably a pair of electrical contacts.To sense the presense of the liquid, DAQ 126 applies a small voltageacross leads 674 and measures the resultant current. As the liquid ofthe microfluidic sample contacts the first and second contacts, thecurrent passing therebetween changes, thereby indicating to DAQ 126 thatthe liquid has arrived at sensor 672.

[0130] Upon recognition that the l;iquid has arrived at sensor 672, theDAQ instructs second actuator 660 to decrease a downstream motive forceacting upon the microfluidic sample 666. For example, DAQ may reduce acurrent flowing through a heat source 676 associated with secondactuator 660 thereby reducing a temperature of a gas therein. Thetemperature reduction reduces the gas pressure acting upon a upstreamportion 678 of microfluidic sample thereby inhibiting the downstreammotion of the microfluidic sample 666. The microfluidic sample ispositioned such that a first portion 680 is located downstream ofactuator 656 and a second portion 682 is located upstream of actuator656.

[0131] To prepare microdroplet 668, DAQ 126 actuates actuator to providea motive force which prepares the microdroplet 668 from the firstportion 680 of microfluidic sample 666. Microdroplet 668 movesdownstream while the second portion 682 of the microfluidic sample 666moves upstream from actuator 656. During microdroplet preparation, valve658 may be closed to substantially isolate the actuator 656 from secondactuator 660 and other upstream portions of the microfluidic device.

[0132] The active positioning element preferably operates as a closedloop element that provides feedback from sensor 672 to the DAQ. Thefeedback is indicated when a microfluidic sample has reached apredetermined position within the microfluidic device. Upon receivingthe feedback, the DAQ changes the state of the actuator providing themotive force to move the microdroplet.

[0133] While the above invention has been described with reference tocertain preferred embodiments, it should be kept in mind that the scopeof the present invention is not limited to these. Thus, one skilled inthe art may find variations of these preferred embodiments which,nevertheless, fall within the spirit of the present invention, whosescope is defined by the claims set forth below.

What is claimed is:
 1. A microfluidic device, comprising: a first gasactuator to provide a gas pressure sufficient to move first samplematerial between first and second spaced apart locations of themicrofluidic device; a second gas actuator to provide a gas pressure tomove second sample material between third and fourth spaced apartlocations of the microfluidic device, the second gas actuator beingspaced apart from the first gas actuator.
 2. The microfluidic device ofclaim 1, wherein the first location is spaced apart from at least one ofthe third and fourth locations.
 3. The microfluidic device of claim 1,wherein the first location is spaced apart from both the third andfourth locations.
 4. The microfluidic device of claim 1, wherein thesecond location overlaps the third location and the second samplematerial comprises at least a portion of the first sample material. 5.The microfluidic device of claim 1, wherein the first location comprisesa first sample processing zone and the first sample material comprisesprocessed sample material prepared at the first sample processing zone.6. The microfluidic device of claim 5, wherein the first sampleprocessing zone is an enrichment zone and the first processed samplematerial comprises enriched sample material.
 7. The microfluidic deviceof claim 5, wherein the first sample processing zone is a cell lysingzone and the first processed sample material comprises intracellularmaterial.
 8. The microfluidic device of claim 5, wherein at least one ofthe second, third, or fourth locations comprises a detection zoneconfigured to obtain data indicative of the presence of a samplematerial.
 9. The microfluidic device of claim 1, wherein the first andsecond gas actuators each comprise a heat source in thermal contact witha volume of gas, whereby actuation of the heat source of a respectivegas actuator causes the gas pressure provided by the gas actuator. 10.The microfluidic device of claim 1, wherein the device comprises asubstrate and the first, second, third, and fourth locations and thefirst and second gas actuators are integral with the substrate.
 11. Themicrofluidic device of claim 1, further comprising a valve disposed toisolate the second gas actuator from the first gas actuator.
 12. Amicrofluidic device for processing a microdroplet of sample, comprising:a first gas actuator to provide a gas pressure sufficient to move themicrodroplet between first and second processing zones of themicrofluidic device; and a second gas actuator to provide a gas pressureto move the microdroplet between the second processing zone and a thirdprocessing zone of the microfluidic device.
 13. The microfluidic deviceof claim 12, wherein the first gas actuator is spaced apart from thesecond gas actuator.
 14. The microfluidic device of claim 13, furthercomprising a valve to isolate the second gas actuator from the first gasactuator.
 15. The microfluidic device of claim 12, wherein the firstprocessing zone is an enrichment zone and the microdroplet comprises anenriched amount of cells.
 16. The microfluidic device of claim 12,wherein the second processing zone is a lysing zone and the microdropletcomprises intracellular material released from cells of the firstmicrodroplet.
 17. The microfluidic device of claim 12, wherein the thirdprocessing zone is a detection zone configured to obtain data indicativeof the presence of a sample substance present in the microdroplet. 18.The microfluidic device of claim 17, wherein the sample substancescomprise polynucleotides.
 19. The microfluidic device of claim 12,wherein the device comprises a substrate and the first, second, andthird locations and the first and second gas actuators are integral withthe substrate.
 20. A method for moving a microdroplet of sample materialwithin a microfluidic device, comprising: providing, at a first locationof the microfluidic device, a first gas pressure sufficient move themicrodroplet between first and second processing zones of themicrofluidic device; and providing, at a second, different location ofthe microfluidic device, a second gas pressure to move the microdropletbetween the second processing zone and a third processing zone of themicrofluidic device
 21. The method of claim 20, wherein the microfluidicdevice comprises a substrate and the first and second gas pressures areprovided by gas actuators that are integral with the substrate.
 22. Themethod of claim 20, further comprising actuating a valve to isolate thesecond processing zone from the first processing zone.
 23. A method formoving a microdroplet of sample material within a microfluidic device,comprising: providing, at a first location of the microfluidic device, afirst gas pressure sufficient to move the microdroplet from a firstmicrodroplet position within the microfluidic device to a secondmicrodroplet position within the microfluidic device; and providing, ata second location of the microfluidic device, a second gas pressure tomove the microdroplet from the second microdroplet position to a thirdmicrodroplet position within the microfluidic device.
 24. The method ofclaim 23, wherein the microfluidic device comprises a substrate and thefirst and second gas pressures are provided by gas actuators that areintegral with the substrate.
 25. The method of claim 22, furthercomprising actuating a valve to isolate the second microdroplet positionfrom the first microdroplet position.
 26. A microfluidic substratecomprising: a microfluidic network, a first gas actuator coupled to saidnetwork at a first location, wherein said first gas actuator, whenactuated, provides gas pressure to move a microfluidic sample within thenetwork, and a second gas actuator coupled to said network at a secondlocation, wherein said second gas actuator, when actuated, provides gaspressure to further move at least a portion of said microfluidic samplewithin said network.
 27. The microfluidic substrate of claim 26 furthercomprising a valve coupled to said network at a third location wherebysaid valve, when closed, substantially isolates the second gas actuatorfrom the first gas actuator.
 28. The microfluidic substrate of claim 26further comprising a microfluidic process zone to receive and processthe microfluidic sample upon actuation of the first gas actuator.